Book/Report

FZJ-2019-00113

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Lipidsynthese in Croynebacterium glutamicum : genetische und biochemische Untersuchungen zu Acyl-CoA Carboxylasen

Tilg, Y. (Corresponding author)FZJ*

2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich

Please use a persistent id in citations: http://hdl.handle.net/2128/21118

Report No.: Juel-3946

Abstract: The Gram-positive soil bacterium C. glutamicum is used for the industrial production of amino acids as L-glutamate and L-lysine. The cell wall of this bacterium and related Corynebacterianeae is in addition to the cytoplasmic membrane built up of a second distinct lipid layer, e. g. the mycolic acid layer. It was shown that these two lipid layers are decisive for the transport of solutes and are likely to influence the efflux of the amino acids. Since the lipid synthesis of the bacterium is not entirely known, the present work describes the isolation of acyl-CoA carboxylases which catalyze the initial reaction of the lipid biosynthesis as well as the characterization of the enzyme activity. In addition to the gene accBC coding for a putative a-subunit of an acyl-CoA carboxylase the four further genes accDA, dtsR1, dtsR2 and dtsR3 were isolated and sequenced. The deduced amino acid sequences of the genes exhibit high identities to ß-subunits of acyl-CoA carboxylases. Enzymatic investigations revealed that AccBC and AccDA built up a functionally enzyme complex. The coexpression of accBC and accDA causes a threefold higher carboxylation of acetyl-CoA to malonyl-CoA, the precursor of the fatty acid biosynthesis, with a specific activity of 161 mU / mg protein. The enzyme carboxylates propionyl-CoA as well. The enzyme is also proved to be essential for C. glutamicum since it is not possible to inactivate accBC or accDA. The inactivation of dtsR1, dtsR2 or dtsR3 led to evident growth limitations, respectively. The growth of each mutant strain is partly restored by supplementation with different fatty acids. This points to a function of the gene products in the lipid biosynthesis. The isolation of the biotinylated AccBC protein by avidin affinity chromatography resulted in the coisolation of proteins identified as DtsR1, DtsR2 and DtsR3. Thereby it is shown that one $\alpha$-subunit can interact with several $\beta$-subunits. In conclusion C. glutamicum could have different acyl-CoA carboxylases whose common $\alpha$-subunit catalizes the CO$_{2}$-fixation and whose $\beta$-subunits are responsible for different yet unknown substrate specifities. The L-glutamate and L-lysine export is influenced by overexpression of the acyl-CoA carboxylase genes. After mutual overexpression of accBC and dtsR1 only 30 % L-glutamate has accumulated. The mutual overexpression of accBC and accDA decreased the L-lysine accumulation by 11 %. Thus the amino acid efflux seems to be correlated with the lipid composition of the membrane.

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Contributing Institute(s):

1. Publikationen vor 2000 (PRE-2000)

Research Program(s):

1. 899 - ohne Topic (POF3-899) (POF3-899)

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